Yujie Wen

Bone marrow transplantation temporarily improves pancreatic function in streptozotocin-induced diabetes: potential involvement of very small embryonic-like cells.

Background. The role of bone marrow (BM)-derived cells in pancreatic β-cell regeneration remains unresolved. We examined whether BM-derived cells are recruited to the site of moderate pancreatic injury and contribute to β-cell regeneration. Methods. Low-dose streptozotocin (STZ) treatment was used to induce moderate pancreatic damage and hyperglycemia. Enhanced green fluorescent protein-positive (EGFP+) BM chimeras were evaluated for β-cell regeneration after STZ treatment. Results. To test the hypothesis that pancreatic tissue injury induces a stromal cell-derived factor (SDF)-1 gradient to chemoattract the stem cells, we evaluated the expression of mRNA for SDF-1 in damaged pancreatic tissue. SDF-1 was significantly increased in the pancreas after damage, peaking at day 10. The majority of BM cells expressing mRNA for pancreatic development markers were detected in the subpopulation of CD45−/Sca-1+/Lin− very small embryonic-like (VSEL) cells. VSEL cells mobilized from BM to peripheral blood in response to pancreatic damage, peaking in peripheral blood at day 5, and were enriched in the pancreas 10 to 15 days after STZ treatment. To confirm a role for BM-derived cells in pancreatic β-cell regeneration, we prepared EGFP+→B6 chimeras. In the EGFP+ chimeras, EGFP+ cells were detected around duct and islets and were positive for insulin after STZ treatment. However, STZ-induced hyperglycemia was reduced only transiently (49–77 days) after pancreatic injury. Conclusions. These data suggest that VSEL cells are mobilized into injured pancreatic tissue and contribute to β-cell regeneration. Transplantation of BM-derived cells improves the function of injured pancreas, although the response is not sufficient to restore sustained normoglycemia.

Stem cell-based strategies for the treatment of Type 1 diabetes mellitus

Importance of the field: β-Cell regeneration and β-cell preservation are two promising therapeutic approaches for the management of patients with type 1 diabetes (T1D). Stem cell-based strategies to address the problems of shortage in β cells, autoimmune and alloimmune responses have become an area of intense study. Areas covered in this review: This review focuses on the progress that has been made in obtaining functional, insulin-producing cells from various types of stem/progenitor cells, including the current knowledge on the immunomodulatory roles of hematopoietic stem cell and multipotent stromal cell in the therapies for T1D. What the reader will gain: A broad overview of recent advancements in this field is provided. The hurdles that remain in the path of using stem cell-based strategies for the treatment of T1D and possible approaches to overcome these challenges are discussed. Take home message: Stem cell-based strategies hold great promise for the treatment of T1D. In spite of the progress that has been made over the last decade, a number of obstacles and concerns need to be cleared before widespread clinical application is possible. In particular, the mechanism of ESC and iPSC-derived β-cell maturation in vivo is poorly understood.

Dissociation between peripheral blood chimerism and tolerance to hindlimb composite tissue transplants: preferential localization of chimerism in donor bone

Background. Mixed chimerism induces donor-specific tolerance to composite tissue allotransplants (CTAs). In the present studies, we used a nonmyeloablative conditioning approach to establish chimerism and promote CTA acceptance. Methods. Wistar Furth (RT1Au) rats were conditioned with 600 to 300 cGy total body irradiation (TBI, day-1), and 100×106 T-cell–depleted ACI (RT1Aabl) bone marrow cells were transplanted on day 0, followed by a 11-day course of tacrolimus and one dose of antilymphocyte serum (day 10). Heterotopic osteomyocutaneous flap transplantation was performed 4 to 6 weeks after bone marrow transplantation. Results. Mixed chimerism was initially achieved in almost all recipients, but long-term acceptance of CTA was only achieved in rats treated with 600 cGy TBI. When anti-αβ-T-cell receptor (TCR) monoclonal antibody (mAb) (day-3) was added into the regimens, donor chimerism was similar to recipients preconditioned without anti-αβ-TCR mAb. However, the long-term CTA survival was significantly improved in chimeras receiving more than or equal to 300 cGy TBI plus anti-αβ-TCR mAb. Higher levels of donor chimerism were associated with CTA acceptance. The majority of flap acceptors lost peripheral blood chimerism within 6 months. However, donor chimerism persisted in the transplanted bone at significantly higher levels compared with other hematopoietic compartments. The compartment donor chimerism may be responsible for the maintenance of tolerance to CTA. Long-term acceptors were tolerant to a donor skin graft challenge even in the absence of peripheral blood chimerism. Conclusions. Mixed chimerism established by nonmyeloablative conditioning induces long-term acceptance of CTA, which is associated with persistent chimerism preferentially in the transplanted donor bone.

Evidence that FoxP3+ regulatory T cells may play a role in promoting long-term acceptance of composite tissue allotransplants.

Background. FoxP3+/CD4+/CD25+ regulatory T cells (Treg) play an important role in maintaining peripheral tolerance and are potent suppressors of T-cell activation. In this study, we evaluated the role of Treg in peripheral tolerance to composite tissue allografts (CTA). Methods. Mixed allogeneic chimeric rats were prepared by preconditioning recipients with anti-&agr;&bgr;-T-cell receptor monoclonal antibody followed by total body irradiation. Animals received T-cell-depleted August Copenhagen Irish bone marrow cells followed by antilymphocyte serum and FK-506. A modified osteomyocutaneous hindlimb flap composed of bone and all limb tissue components was placed in animals with chimerism greater than or equal to 1% on day 28. Recipients with CTA surviving more than or equal to 6 months were evaluated for Treg. Skin samples from tolerant long-term allogeneic transplanted, syngeneic transplanted, rejected, and naïve animals were immunostained with fluorochrome-conjugated anti-FoxP3 and anti-CD4 monoclonal antibody and visualized under a laser confocal microscope. Results. Significant CD4+/FoxP3+ Treg infiltrates were observed in tolerant donor-allograft skin samples. No graft infiltrating FoxP3+ cells were observed in rejector, naïve, or skin from syngeneic CTA. In parallel experiments, mixed leukocyte reaction assays were performed to investigate the suppressor function of Treg cells. Splenocytes from tolerant, rejected, and naïve rats were sorted by flow cytometry for CD4+/CD25+ T cells. Treg demonstrated similar suppressive levels between the three groups. Conclusions. These data suggest that Treg may play an important role in maintenance of tolerance and promoting graft acceptance in long-term CTA acceptors and may explain the favorable outcomes observed in clinical CTA recipients.

Sensitized Recipients Exhibit Accelerated but not Hyperacute Rejection of Vascularized Composite Tissue Allografts

Background. Currently, the donor-recipient matching process for vascularized composite tissue allotransplantation (VCTA) closely follows the standard practices for solid organ transplantation. Sensitization is considered a contraindication to VCTA. However, the role of sensitization in VCTA rejection is largely unstudied. Methods. Major histocompatibility-mismatched ACI (RT1a) donors and Wistar Furth (WF) (RT1u) recipients were used to determine whether sensitization would lead to hyperacute rejection in VCTA as in other organs, such as kidneys. WF rats were presensitized to ACI antigens by skin transplantation and received heterotopic osteomyocutaneous VCTA flaps. Kidney transplants served as controls. Results. Production of anti-donor antibody was detected in WF recipients after rejection of the ACI skin grafts. Sensitized WF rats rejected VCTA grafts from ACI rats significantly faster (P<0.05) than unsensitized recipients, but not hyperacutely. Rejection in the sensitized recipients was not prevented by immunosuppression with FK506 and mycophenolate mofetil. In contrast, kidney allografts from ACI rats were hyperacutely rejected within 30 min by sensitized recipients. To confirm the role of antibody-mediated rejection in the sensitized recipients, serum from presensitized rats was adoptively transferred into naïve WF rats. Hyperacute rejection occurred only in transplanted kidneys but not VCTA. Histologic examination of tissues from acceleratedly rejected VCTA showed dense lymphocytic infiltrates, and no antibody deposition. Conclusions. VCTA are rejected in an accelerated fashion but not hyperacutely in the presence of allosensitization and preformed anti-donor antibody. The rejection of VCTA in sensitized recipients is mainly cell mediated and differs mechanistically from that for renal transplants.

DOCK2 Is Critical for CD8+TCR− Graft Facilitating Cells to Enhance Engraftment of Hematopoietic Stem and Progenitor Cells

CD8+TCR− graft facilitating cells (FCs) enhance engraftment of hematopoietic stem cells (HSCs) in allogeneic and syngeneic recipients. The mechanisms by which FCs promote HSC engraftment and tolerance induction have not been fully elucidated. Here, we provide data to support a critical role for dedicator of cytokinesis 2 (DOCK2) in multiple aspects of FCs function. DOCK2−/− FCs exhibit compromised facilitative function in vivo as evidenced by the loss of engraftment‐enhancing capability for c‐Kit+Sca‐1+lineage− (KSL) cells, and compromised ability to promote KSL cell homing and lodgment in hematopoietic niche. Deletion of DOCK2 abrogates the ability of FCs to induce differentiation of naïve CD4+CD25− T cells into FoxP3+ regulatory T cells and interleukin‐10‐producing type 1 regulatory T cells in vitro. Moreover, DOCK2−/− FCs are unable to promote survival of KSL cells when cocultured with KSL cells. DOCK2−/− FCs also exhibit compromised migration to stroma‐derived factor‐1 in vitro and impaired homing to the bone marrow in vivo. In conclusion, our results demonstrate that DOCK2 is critical for FCs to maintain its immunomodulatory function and exert its trophic effects on KSL cells. These findings may have direct clinical relevance to promote HSC engraftment for treatment of autoimmunity, hemoglobinopathies, and to induce transplantation tolerance. Stem Cells 2014;32:2732–2743

A critical role for the TLR4/TRIF pathway in allogeneic hematopoietic cell rejection by innate immune cells.

We show for the first time that signaling through the TLR4/TRIF pathway plays a critical role in allogeneic bone marrow cell (BMC) rejection. This appears to be unique to BMCs as organ allografts are rejected mainly via MyD88 signaling. Using T- or T-/B-cell-deficient mice, we found that BMC allorejection occurred early before T-cell activation and was T- and B-cell independent, suggesting an effector role for innate immune cells in BMC rejection. We further demonstrated the innate immune signaling in BMC allorejection by showing superior engraftment in mice deficient in TRIF or TLR4 but not in MyD88 or TLR3. The restored cytotoxicity in TRIF-deficient recipients transferred with wild-type F4/80+ or NK1.1+ cells suggests TRIF signaling dependence on macrophages or NK cells in early BMC rejection. Production of the proinflammatory cytokine IL-6 and TRIF relevant chemokine MCP-1 was significantly increased early after bone marrow transplantation. In vivo specific depletion of macrophages or NK innate immune cells in combination with anti-CD154/rapamycin resulted in additive-enhanced allogeneic engraftment. The requirement for irradiation was completely eliminated when both macrophages and NK cells were depleted in combination with anti-CD154/rapamycin to target T- and B-cells, supporting the hypothesis that two barriers involving innate and adaptive immunity exist in mediating the rejection of allogeneic BMCs. In summary, our results clearly demonstrate a previously unappreciated role for innate immunity in BMC allorejection via signaling through a unique MyD88-independent TLR4/TRIF mechanism. These findings may have direct clinical impact on strategies for conditioning recipients for stem cell transplantation.

Characterization of Human CD8(+)TCR(-) Facilitating Cells In Vitro and In Vivo in a NOD/SCID/IL2rγ(null) Mouse Model.

CD8+/TCR− facilitating cells (FCs) in mouse bone marrow (BM) significantly enhance engraftment of hematopoietic stem/progenitor cells (HSPCs). Human FC phenotype and mechanism of action remain to be defined. We report, for the first time, the phenotypic characterization of human FCs and correlation of phenotype with function. Approximately half of human FCs are CD8+/TCR−/CD56 negative (CD56neg); the remainder are CD8+/TCR−/CD56 bright (CD56bright). The CD56neg FC subpopulation significantly promotes homing of HSPCs to BM in nonobese diabetic/severe combined immunodeficiency/IL‐2 receptor γ‐chain knockout mouse recipients and enhances hematopoietic colony formation in vitro. The CD56neg FC subpopulation promotes rapid reconstitution of donor HSPCs without graft‐versus‐host disease (GVHD); recipients of CD56bright FCs plus HSPCs exhibit low donor chimerism early after transplantation, but the level of chimerism significantly increases with time. Recipients of HSPCs plus CD56neg or CD56bright FCs showed durable donor chimerism at significantly higher levels in BM. The majority of both FC subpopulations express CXCR4. Coculture of CD56bright FCs with HSPCs upregulates cathelicidin and β‐defensin 2, factors that prime responsiveness of HSPCs to stromal cell–derived factor 1. Both FC subpopulations significantly upregulated mRNA expression of the HSPC growth factors and Flt3 ligand. These results indicate that human FCs exert a direct effect on HSPCs to enhance engraftment. Human FCs offer a potential regulatory cell‐based therapy for enhancement of engraftment and prevention of GVHD.

Fms‐Like Tyrosine Kinase 3‐Ligand Contributes to the Development and Function of the Subpopulation of CD8α+ Plasmacytoid Precursor Dendritic Cells in CD8+/TCR− Facilitating Cells

Facilitating cells (FC) are a CD8+TCR− bone marrow subpopulation that enhance engraftment of purified hematopoietic stem cells (HSC) and induce antigen‐specific CD4+CD25+FoxP3+ regulatory T cell (Treg) in vivo. The major subpopulation in FC resembles plasmacytoid precursor dendritic cells (p‐preDC) both phenotypically and functionally. Here, we report that the number of FC was significantly reduced in Fms‐like tyrosine kinase 3‐ligand‐knockout (Flt3‐L‐KO) mice. Specifically, there was a selective decrease in the B220+CD11c+CD11b− p‐preDC FC subpopulation. The p‐preDC FC subpopulation in FC total is restored after Flt3‐L administration to Flt3‐L‐KO mice. FC from Flt3‐L‐KO donors exhibit impaired facilitation of allogeneic HSC engraftment in ablatively conditioned mice (B6 → NOD) as well as in mice conditioned with reduced intensity conditioning (B6 → BALB/c). In addition, the number of CD4+CD25+Foxp3+ Treg from Flt3‐L‐KO mice is significantly decreased. This was associated with the expression of chemokine receptor CXCR3+ or CCR5+ on Treg. Treg from the spleen of Flt3‐L‐KO mice showed impaired facilitation of engraftment of allogeneic HSC compared to wild‐type Treg. Flt3‐L treatment significantly expanded Treg, and restored their facilitating function. These results suggest that Flt3‐L is an important growth factor in the development and homeostasis of p‐preDC FC and in the role of FC inducing generation of Treg. Flt3‐L provides potent immunoregulatory properties that may be clinically useful to improve tolerance induction and enhance the function of allogeneic cell therapies. Stem Cells 2018;36:1567–1577

Facilitating cells: Translation of hematopoietic chimerism to achieve clinical tolerance

ABSTRACT For over 50 y the association between hematopoietic chimerism and tolerance has been recognized. This originated with the brilliant observation by Dr. Ray Owen that freemartin cattle twins that shared a common placental blood supply were red blood cell chimeras, which led to the discovery that hematopoietic chimerism resulted in actively acquired tolerance. This was first confirmed in neonatal mice by Medawar et al. and subsequently in adult rodents. Fifty years later this concept has been successfully translated to solid organ transplant recipients in the clinic. The field is new, but cell-based therapies are being used with increasing frequency to induce tolerance and immunomodulation. The future is bright. This review focuses on chimerism and tolerance: past, present and prospects for the future.